Construction and identification of fully synthetic nanobody phage display library

• 1、Tianjin University Science and Technology,College of Biotechnology,TianJin 300457

Abstract：Objective: On the basis of the M13 phage display technology platform, a fully synthetic nanoantibody display library was constructed in order to develop various nanoantibodies with potential therapeutic and diagnostic applications.Methods: The number and sequence of amino acids in the skeletal region and the CDRs region of the fully synthetic nanoantibody library were determined by comparing the large-scale sequencing of the natural nanoantibody library and the sequencing results. Nanobody skeleton sequence as a template for three CDRs area design degenerate primers, NNK randomized methods introduced random mutations, overlap extension PCR to obtain VHH gene library, to build it to phagocytosis pCantab 5E carrier, and then use the electricity conversion to convert carrier to E. coil TG1 to build primary phage antibody library, after helper phage to save the phage display library, finally analyze the diversity and capacity of phage display libraries and identification. Results: The total size of the phage display library is 1 × 107 colony-forming units (CFU). The insertion rate of the fully synthetic nanobody library was about 73.3%. The library has a high diversity.. Conclusion: the fully synthetic nanoantibody library was successfully constructed with good insertion rate and diversity, which provides material and technical support for the preparation of nanobodies with therapeutic and diagnostic functions.

Keywords： Nanobody Phage display Full synthetic library Construction Iditification